Evaluation of apparent formation constants of pentacyclic triterpene acids complexes with derivatized β- and γ-cyclodextrins by reversed phase liquid chromatography

doi:10.1016/j.chroma.2004.06.133

Journal of Chromatography A

Volume 1049, Issues 1–2, 17 September 2004, Pages 37-42

https://doi.org/10.1016/j.chroma.2004.06.133 Get rights and content

Abstract

A reversed phase HPLC method has been investigated in order to resolve three main pentacyclic triterpene acids (oleanolic-, betulinic- and ursolic acid) found in a lot of plants. Some of them (oleanolic and ursolic acids) are position isomers and their resolution is highly improved by the addition of derivatized cyclodextrins in mobile phase. The formation of 1:1 inclusion complexes was assumed. Apparent formation constants of triterpene acids with DM-β-CD and HP-γ-CD were determined by HPLC method. Experimental results confirmed the complexation model and explained the modification of elution order according to the type of cyclodextrin added to the mobile phase. The influence of mobile phase organic modifier on apparent formation constants was also investigated. Results proved the competition between cyclodextrins hydrophobic cavity and organic solvent towards triterpene acids affinity.

Introduction

Triterpenoids, such as oleanolic, ursolic or betulinic acids are pentacyclic molecules (Fig. 1). As secondary metabolites of some plants, they possess pharmacological properties. The triterpene acids have been shown to exhibit significant anti-HIV activity [1].

Betulinic acid can act as a selective inhibitor of human melanoma in cell culture and animal models that function by induction apoptosis, whereas other compounds currently used in cancer therapy (taxol, vinblastine) inhibit the replication of both cancer and normal cells [2]. Ursolic acid has also shown significant cytotoxicity in lymphocytic leukemia cells [3]. As this molecule is relatively non-toxic and possesses anti-inflammatory and antihyperlipidemic properties, it has been used in cosmetics. Oleanolic acid has also been proposed as an anti-inflammatory and antiarthritic agent.

Oleanolic and ursolic acids occur especially in the waxy coatings of leaves and on fruits such as apple and pear. Betulinic acid can be found in birch, plane and cork barks [4], [5]. Betulinic, oleanolic and ursolic acids have been identified in almond hulls [6].

The most common analytical methods of triterpenoids in plants are liquid chromatography [7], [8], [9], gas chromatography after silylation [10], [11] or methylation [6]. The identification of betulinic, oleanolic and ursolic acids in natural extracts has been performed by GC–MS after derivatisation [4], [12], [13]. Capillary supercritical fluid chromatography [14] and cyclodextrin-modified micellar electrokinetic chromatography [15] have also been used for plant analysis.

Liquid chromatography–electrospray mass spectrometry (LC–ESI-MS) has already been used to identify betulinic acid [7] and liquid chromatography–atmospheric pressure chemical ionisation mass spectrometry (LC–APCI-MS) to quantify ursolic acid [16], as triterpene acids have weak chromophores.

The resolution of oleanolic and ursolic acids by LC seems difficult on reversed phase as these molecules are position isomers. The addition of cyclodextrins (CD) to the mobile phase was therefore investigated to improve the separation. In fact, cyclodextrins have already been used as mobile phase modifiers in HPLC applications for the analysis of steroids [17].

Cyclodextrins (CDs, α, β, γ) are torus-shaped, naturally occurring, enzymatically synthesized, cyclic oligosaccharides composed of six to nine α-1,4 linked d-glucopyranose units per molecule (α-, β-, γ- and δ-cyclodextrin, respectively). While the exterior of the molecule is hydrophilic, its hydrophobic cavity may selectively include molecules with appropriately sized organic compounds by forming non-covalent inclusion complexes. Besides, the internal cavity being less polar than the surrounding water molecules, chemical properties of the guest, once included, may be dramatically affected. For the following of this study, it is interesting to note the slight solubility of cyclodextrins in water and more particularly in organic solvent–water mixtures. Our study was therefore performed with cyclodextrins derivatized by dimethyl- or hydroxypropyl groups.

In the present paper, a simple and fast high-performance liquid chromatographic method for the separation of the most common triterpene acids is reported. The use of cyclodextrins as HPLC modifier has never been reported in the literature for the separation of triterpene acids. The resolution of these closely related hydrophobic triterpene acids requires an HPLC mobile phase containing a high percentage of acetonitrile and one derivatized cyclodextrin to obtain sufficient selectivity. Thus the selectivity was assessed by the addition of dimethyl-β or hydroxypropyl-γ-cyclodextrin to the mobile phase to promote hydrophobic interactions in order to differentiate structurally similar molecules.

The aim of our research was also to assess the triterpene acid–cyclodextrin inclusion complex by HPLC. Indeed, cyclodextrins are known to improve some properties of drugs (solubility, bioavailability) and enhance drug activity by encapsulation of the active molecule. The stoichiometry of the complex and the corresponding apparent formation constant (K_f) have been determined from the change in retention factors as the concentration of cyclodextrin in the mobile phase varied. This paper deals with the choice of a cyclodextrin for a C₁₈ stationary phase. Complex apparent formation constants (CD-triterpene acid) have been determined experimentally at several compositions of the mobile phase.

Section snippets

Apparatus

The HPLC system used was a Varian model 9012 ternary pump (Les Ulis, France), equipped with a Rheodyne (Cotati, CA, USA) model 7125 injection valve fitted with a 20 μL injection loop. The triterpene acids possess a UV absorption maximum at 225 nm, so this wavelength was used for detection. A Shimadzu SPD-6A spectrophotometric detector was connected to a computer equipped with EZChrom-Elit software version 2.5 (Scientific Software Inc., Pleasanton, CA, USA). A C₁₈ silica Lichrospher^® 100 RP-18

Results and discussion

Acetonitrile–phosphate buffer (pH 3.5) was selected as the mobile phase because of the good elution of the triterpene acids without cyclodextrins. With methanol as organic modifier in mobile phase, the cyclodextrins solubility is improved but the retention of triterpene acids was dramatically increased and a collapse of chromatographic peaks was observed.

Conclusion

The separation of triterpene acids by HPLC was improved by addition of cyclodextrins (DM-β-CD, HP-γ-CD) to the acetonitrile–phosphate buffer mobile phase. The formation of inclusion complexes between triterpene acids and cyclodextrins explains the selectivity modification and the elution order of analytes. The stability of these complexes depends on the size and conformation of triterpene acids as well as on the hydrophobic cavity size of cyclodextrins.

Values of the complex apparent formation

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Evaluation of apparent formation constants of pentacyclic triterpene acids complexes with derivatized β- and γ-cyclodextrins by reversed phase liquid chromatography

Abstract

Introduction

Section snippets

Apparatus

Results and discussion

Conclusion

Ind. Crops Products

J. Chromatogr. B

Pharmaceut. Biomed. Anal.

Eur. J. Pharmaceut. Sci.

Microchem. J.

J. Ethnopharmacol.

J. Chromatogr. A

J. Chromatogr. A

J. Chromatogr. A

Mini Rev. Med. Chem.